Results are presented which demonstrate that purified trisialoganglioside spontaneously incorporates into preformed phospholipid vesicles. Determinations of the extent of incorporation were made by separating large unilamellar dipalmitoylphosphatidylcholine vesicles containing incorporated ganglioside from micellar ganglioside on a Sepharose-2B column. Incorporation occurs without appreciably altering the vesicular character of the phospholipid bilayer as judged by the maintenance of an outside/inside ratio, determined by 31P NMR, comparable to that of the original vesicles. All of the incorporated ganglioside is accessible to neuraminidase, indicating that incorporation occurs only on the outer face of the bilayer. The thermotropic behavior of these asymmetric dipalmitoylphosphatidylcholine-trisialoganglioside vesicles, examined by high sensitivity scanning calorimetry, strongly suggests that the incorporated ganglioside is intercalated into the outer monolayer of the vesicle bilayer. Calorimetric studies indicate that the ganglioside stabilizes these vesicular structures by inhibiting the fusion of small vesicles that occurs below the phase-tranisition temperature. These structures are a representative model system, which like the mammalian plasma membrane contain an asymmetric distribution of glycosphingolipid in the outer surface.
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